Overexpression of initiation factor eIF-4E does not relieve the translational repression of ribosomal protein mRNAs in quiescent cells.

Abstract

Translation of ribosomal protein (rp) mRNA is selectively repressed in mouse erythroleukemia (MEL) cells, which cease to proliferate upon differentiation, and in NIH 3T3 cells, for which growth is arrested by either serum starvation, contact inhibition, or treatment with the DNA polymerase inhibitor, aphidicolin. The efficiency of translation of rp mRNAs correlates with the expression of the gene encoding the cap binding protein, eIF-4E, as indicated by the fact that the abundance of the corresponding mRNA and protein also fluctuates in a growth-dependent manner. To examine the hypothesis that eIF-4E plays a role in regulation of the translation efficiency of rp mRNAs, we utilized an NIH 3T3-derived eIF-4E-overexpressing cell line. These cells overproduce eIF-4E to the extent that even under conditions of growth arrest, the abundance of the respective protein in its active (phosphorylated) form is higher than that found in exponentially growing NIH 3T3 cells. Nevertheless, this surplus amount of eIF-4E does not prevent the translational repression of rp mRNAs when the growth of these cells is arrested by blocking DNA synthesis with aphidicolin or hydroxyurea. In complementary experiments we used an in vitro translation system to compare the competitive potential of mRNAs, containing the translational cis-regulatory element (5' terminal oligopyrimidne tract) and mRNAs lacking such a motif, for the cap binding protein. Our results demonstrate that both types of mRNAs, regardless of their translational response to growth arrest, exhibit similar sensitivity to the cap analogue m7G(5')ppp(5')G. It appears, therefore, that the presence of the regulatory sequence at the 5' terminus of rp mRNAs does not lessen its competitive potential for the cap binding protein and that the growth-dependent decrease in the activity of eIF-4E does not play a key role in the repression of translation of rp mRNAs.

The abundance as well as the synthesis rate and steady-state level of the encoded protein in growing and resting cells, (a) Fluctuations in the relative abundance of eIF-4E mRNA during transitions between growing and resting states. Poly(A)+ mRNA was isolated () from untreated (C) or HMBA treated for 72 h (H) MEL cells (MELC); from NIH 3T3 cells, which were either exponentially growing in 10% serum (10), serum starved (.5), or serum stimulated (Re); and from lymphosarcoma cells (P1798), which were either untreated (C), dexamethasone treated for 24 h (D), or 24 h after hormone withdrawal (W). Poly(A)+ mRNA from MEL cells (0.5 μg), P1798 cells (0.5 μg), and from NIH 3T3 cells (2 μg) was analyzed by Northern blot hybridization with eIF-4E probe. Only the two most intense eIF-4E transcripts (1.8 and 1.6 kb long), out of the five normally visualized by this probe (), are presented here, (b) Relative synthesis rate of eIF-4E. Untreated (C) sparsely cultured NIH 3T3 cells and cells treated with 5 μg/ml aphidicolin for 24 h (A) were labeled for 4 h with [35S]methionine and labeled eIF-4E was immunoprecipitated from cell lysates containing equal trichloroacetic acid-precipitable radioactivity. The immunoprecipitates were analyzed by SDS-PAGE as described in . The eIF-4E band (identified by its mol.wt.) is indicated, (c) Steady-state level of eIF-4E protein. Cellular proteins (38 μg) from untreated (C) and dexamethasone-treated (D) P1798 cells (C) were analyzed by Western immunoblotting as described in . The bands representing eIF-4E and actin are indicated.

Overexpression of eIF-4E in growing and nongrowing NIH 3T3 cells, (a) Relative abundance of eIF-4E mRNA. Cytoplasmic RNA was extracted from NIH 3T3 cells (NIH) or P2 cells (P2), which were either untreated (C) or aphidicolin treated (A). These RNAs (10 μg) were analyzed by Northern blot hybridization with eIF-4E probe. The ∼5.0-kb transcript apparent in the transfected cells probably reflects readthrough of the polyadenylation signal in the eIF-4E cDNA and termination within the 3′ LTR of the Moloney murine leukemia virus sequence (). The ethidium bromide-stained 28S rRNA served as an internal reference standard for showing that equal amounts of RNA were loaded on each lane, (b) eIF-4E protein levels. Cellular proteins (38 μg) from aphidicolin-treated (Aph) P2 cells (P2) and untreated NIH 3T3 cells (NIH) were analyzed by Western immunoblotting and detection by ECL as described in . The bands representing eIF-4E and actin are indicated. (c) Phosphorylation state of eIF-4E. Cellular proteins (120 μg) from aphidicolin-treated (Aph) P2 cells (P2) and untreated NIH 3T3 cells (NIH) were analyzed by two-dimensional IEF/SDS-PAGE Western immunoblotting and detection by ECL as described in . The sections of the gels containing actin as well as the phosphorylated (4E-P) and unphosphorylated (4E) forms of eIF-4E are shown in the autoradiograms. The relative position of these proteins and the direction of migration in each dimension are depicted in the scheme at the left. The relative abundance of eIF-4E is presented at the right of (b) and (c). It was determined by densitometric scanning of the respective bands in (b) and of the spots representing the phosphorylated and unphosphorylated forms of the protein in (c). The densitometric signals of eIF-4E were normalized to that of actin in the respective autoradiograms. The values obtained for the growing NIH 3T3 cells were used as a reference, which was arbitrarily set at 1.0, whereas those derived for the aphidicolin-treated P2 cells were normalized to the reference value.

The translation of rp mRNAs is repressed upon growth arrest of eIF-4E-overexpressing cells. Cytoplasmic extracts from P2 cells that were either untreated (Con) or growth arrested (+) by 24-h treatment with either aphidicolin or hydroxyurea, or 24 h after withdrawal of the drug (W), were centrifuged through sucrose gradients and separated into polysomal (P) and subpolysomal (S) fractions. Poly(A)+ mRNA from equivalent aliquots of these fractions was analyzed by Northern blot hybridization with the probes indicated at the left. Because of variations in the amount of cytoplasmic extracts separated in each gradient and different exposure times for the + or W lanes, even for the same drug or the same probe, quantitative comparisons of the autoradiographic signal can be made only between the polysomal and subpolysomal fractions of the same gradient. The percentage of mRNA in polysomal fractions was assessed as described in , and the results presented at the right are an average of the number of experiments indicated in parentheses. The SEM in all experiments with three or more measurments was less than 10%. The lower band in actin-probed lane S of hydroxyurea-withdrawal cells is a contamination and not a fast running actin mRNA.

The effect of the cap analogue, m7G(5′)ppp(5′)G, on the translation efficiency of 5′ TOP-containing and -lacking mRNAs. (a) Dependence of the amount of hGH synthesized in rabbit reticulocyte lysate on the amount of total poly(A)+ RNA used in the translation system. Increasing amounts of total poly(A)+ RNA from NIH 3T3 cells overexpressing S16wt(1–29)-GH were translated in reticulocyte lysate and the amount of hGH synthesized was measured by radioimmunoassay, as described in . The conversion of radioactivity data into mass was based on radioimmunoassay of hGH standards. (b,c) The effect of increasing concentrations of the cap analogue, m7G(5′)ppp(5′)G, on the translation efficiency of total proteins and hGH mRNA with various 5′ termini. Poly(A)+ RNA (100, 20, 45, and 100 ng) from NIH 3T3 cell lines expressing mRNA encoding: Act(73)-GH (Actin), S16CM3-GH (CM3), S16wt(1–29)-GH (rpS16), and L13a-GH (rpL13a) (; ), respectively, were translated in rabbit reticulocyte lysate in the absence or presence of increasing concentrations of the cap analogue. The synthesis of total proteins (dashed line) was monitored by measuring the incorporation of [35S]methionine into trichloroacetic acid-precipitable material and represents an average of numerous experiments. The amount of the hGH synthesized (solid lines) was monitored by radioimmunoassay. In both cases, the results were normalized to the value obtained in the absence of cap analogue and are presented as averages ± SEMs of at least three independent experiments. The reference values were set at 100% and they represent 23, 70, 60, and 80 pg of hGH produced during 90-min incubation with poly(A)+ RNAs listed above, respectively. The variation in the level of synthesized hGH reflects primarily the difference in the abundance of hGH mRNA in each RNA preparation. The sequences depicted in the figure represent the 5′ terminal sequences of the respective transcripts ().